Adipose-derived stromal cells (ASCs) have the capacity to undergo osteoblastic differentiation. These postnatal cells may be used to design cell-based regenerative therapies for skeletal repair in debilitating genetic disorders, degenerative diseases, and traumatic or post-surgical tissue deficits. While we have made substantial progress in studying the cell biology underlying osteodifferentiation of ASCs, we are limited by working with a heterogeneous cell population, and specific enriched, subpopulations of osteoprogenitor cells with the greatest osteopotential need to be identified. Current investigations utilize heterogeneous cells and have several disadvantages. Subpopulation gene expression changes may be attenuated, or even completely masked, due to the presence of multiple cell types. Determining whether there is a single set of progenitor cells capable of osteodifferentiation would be extremely beneficial, both in terms of advancing our knowledge of postnatal multipotent cells and for optimizing clinical therapeutic strategies. Preliminary data suggest that it is indeed possible to enrich heterogeneous ASCs for osteoprogenitors using fluorescent-activated cell sorting (FACS). The first Specific Aim focuses on performing cell fractionation experiments on primary cultures of cells derived from inguinal fat pads of adult mice. Cells will be subdivided according to expression of individual candidate markers (i.e. marker+ vs. marker-) previously identified as markers of adult stem cells. These cells will be assayed in vitro for their potential to proliferate and differentiate down the osteogenic lineage compared to unfractionated ASCs. In the second Specific Aim, the ASC subpopulations identified in the first Specific Aim with greatest osteopotential will be placed in an in vivo mouse calvarial defect model to determine if they enhance skeletal regeneration. Together, these Aims will use flow-cytometry in concert with both in vitro and in vivo osteoblastic differentiation assays to prospectively delineate the cell-surface marker profile of osteoprogenitors residing within the heterogeneous mix of harvested ASCs. Skeletal disease poses a serious challenge to a variety of medical disciplines. At the same time, it also poses a significant socioeconomic burden on our healthcare infrastructure exceeding $1 billion annually. Current clinical approaches to regeneration of the skeleton include use of autogenous grafts, allogenic materials and prosthetic products, each with its own inherent disadvantages. The ability to harness the regenerative potential of cellular building block progenitor cells portends an exciting and novel paradigm for management of skeletal disease.